Long range intercellular communication among astrocytes is in large part mediated by gap junction channels through which ions and metabolites pass directly from one cell to the next. The pore of astrocyte gap junctions is formed primarily of the gap junction protein connexin43. Recent evidence indicates that other proteins are associated with Cx43 at gap junctions, and we have termed this macromolecular complex the Nexus. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction. In order to test this hypothesis, we will determine the identities of the other proteins that bind to Cx43 in astrocytes and in transfected cells, implement high throughput and quantitative methods to identify novel Nexus proteins, measure the strengths of interaction, and perform physiological experiments with Cx43 mutants and with Cx43 binding partners to determine the functional consequences of such interactions. In addition, a major goal of this application is to obtain structural information regarding domains of Cx43 that interact with each other and with other proteins. These studies use a multidisciplinary approach directed at exploring a new concept in the field and as such are expected to lead to novel understanding of roles that gap junctions play in the nervous system and elsewhere.